Luciferins

ABSTRACT

Novel luciferins, methods of making luciferins, and uses of the same are disclosed.

CLAIM OF PRIORITY

This application is a divisional of U.S. patent application Ser. No.12/040,812, filed on Feb. 29, 2008, now U.S. Pat. No. 7,910,087 whichclaims the benefit of U.S. patent application Ser. No. 60/904,731, filedon Mar. 2, 2007. The entire contents of the foregoing are herebyincorporated by reference.

TECHNICAL FIELD

This invention relates to luciferins, methods of making luciferins, andto uses of the same.

BACKGROUND

Firefly luciferase is widely used for bioluminescent imaging in mice.However, when combined with firefly luciferin, the emitted yellow-greenlight (560 nm) penetrates poorly through tissue due to absorption byhemoglobin and Rayleigh scattering. For optimal bioluminescent imaging,longer wavelengths (>650 nm) would be desirable.

Some mutants of firefly and click beetle luciferases maximally emitlight as high as 615 nm (Anal. Biochem., 2005, 345(1):140), and railroadworm luciferase naturally emits light at 623 nm (Biochemistry, 1999,38(26):8271). Most of these red-shifted luciferases, however, have notbeen well characterized, and for those examples that have, thebathochromatic shift in emission is concomitant with a substantial lossin light output, and often a significant loss of affinity for bothluciferin and ATP.

Referring to FIG. 1, enzymatic oxidation of firefly luciferin (1) withfirefly luciferase (LUC), and subsequent decarboxylation, generatesoxyluciferin (described by (1′A) and (1′B)) in an electronically-excitedstate (FIG. 1). This molecule returns to the electronic ground state byemitting a photon with very high quantum yield (0.9) (see, e.g., Arch.Biochem. Biophys., 88 (1960) 136-141). The wavelength of the emittedphoton is determined by the structure and electronic properties of theoxyluciferin chromophore within the luciferase binding pocket. Atphysiological pH, the emission wavelength of wild-type fireflyluciferase is 560 nm. At low pH (˜6), this emission is red-shifted to ashigh as 617 nm, but with a decreased quantum yield.

SUMMARY

Generally, luciferins, e.g., N-substituted amino luciferins, such asN-alkylamino luciferins, or salts or derivatives thereof are disclosed,as well as methods of use thereof. These new luciferins are substratesfor luciferases, i.e., they emit light when combined with a luciferase.

In one aspect, the invention features compounds of Structure (I), orsalts or acid esters thereof.

In Structure (I), R₁ and R₂ are each independently H (provided that R₁and R₂ are not both H), a first moiety including up to 12 carbon atoms,or a first moiety including a near infrared fluorophore. R₃ is H, OH, asecond moiety including up to 12 carbon atoms, or a second moiety thatincludes a near infrared fluorophore. R₄ and R₅ are each independentlyH, OH, or a moiety that includes up to 6 carbon atoms. R₆ and R₇ areeach independently H, or a moiety including up to 8 carbon atoms. R₁,R₂, R₃, or R₅ may together with one or more of its immediate neighborsdefine one or more ring systems, each including up to 14 carbon atoms.

In some embodiments, the first and/or second moiety including up to 12carbon atoms also includes one or more N, O, P, S, F, Cl, Br, or I.

The moieties that include up to 6 carbon atoms and/or the moieties thatinclude up to 8 carbon atoms can also include one or more N, O, P, S, F,Cl, Br, or I.

The first and/or second moieties that include the near infraredfluorophore can also include a spacer including up to 24 carbon atoms,or a polymer fragment, e.g., a polymer fragment of a water-solublepolymer such as a polyethylene glycol or a copolymer thereof. The spacercan also include one or more N, O, P, S, F, Cl, Br, or I.

For example, the one or more defined ring systems can further includeone or more N, O, P, S, F, Cl, Br, or I.

In particular embodiments, R₃, R₄, R₅, R₆, and R₇ are each hydrogen, oran alkyl group, e.g., one having fewer than 6 carbon atoms, or havingfewer than 4 carbon atoms.

In some embodiments, R₁ and R₂ together define a ring, the compoundsbeing represented by Structure (II), which is shown below.

In some embodiments, R₁ and R₅ together define a ring, the compoundsbeing represented by Structure (III). Such compounds are characterizedas having hindered rotation about the Ar—N(R₁R₂) bond of the ofStructure (III). Rotation can be further hindered, e.g., by having acarbon-carbon double bond in the ring. A double bond may also provideadditional conjugation with the π-system of the chromophore.

In other embodiments, R₂ and R₃ together define a ring, the compoundsbeing represented by Structure (IV). Such compounds are characterized ashaving hindered rotation about the Ar—N(R₁R₂) bond of the of Structure(IV).

In certain embodiments, R₁ and R₅ and R₂ and R₃ together define a ring,the compounds being represented by Structure (V). Such compounds arecharacterized as having extremely hindered rotation about the Ar—N(R₁R₂)bond of the of Structure (V). Rotation can be further hindered, e.g., byhaving one or more carbon-carbon double bonds in one or more rings.

In some instances, the one or more ring systems described above candefine a 5, 6, and/or 7-membered rings.

In some implementations, R₁ and/or R₂ and/or R₃ comprise a near infraredfluorophore.

In specific implementations, the compounds of Structure (I) arerepresented by Structure (VII).

In such instances, R₁ and/or R₂ can be, e.g., alkyl groups, such asmethyl groups.

The salts of any of the luciferins described herein can be, e.g.,lithium, sodium, potassium, calcium, magnesium or ammonium salts (e.g.,trialkylammonium salts). The esters can be, e.g., NHS esters, alkylesters (e.g., C1-C3 alkyl esters), phenyl esters, benzyl esters oradenosine monophosphate (AMP) esters.

In another aspect, the invention features N-alkyl luciferins, or saltsor acid ester thereof. For example, the N-alkyl luciferin can be amono-alkyl luciferin or a di-N-alkyl luciferin.

In a specific embodiment, the N-alkyl luciferin has Structure (1a),which is shown below.

In some embodiments, when R₁ is not H, R₂ is a substrate for a protease(e.g., an amino acid residue or polypeptide). In some embodiments, thecarboxyl group is covalently bound to a protecting group. See, e.g.,U.S. Pat. Nos. 5,035,999 and 7,148,030.

In another aspect, the invention features methods of generating lightthat include providing a luciferase having a binding pocket sized forany luciferin described herein (or an equivalent thereof, e.g., a saltor ester thereof); and combining the luciferin with the luciferase. Theluciferase can be a wild-type luciferase, such as a firefly, clickbeetle, or railroad worm luciferase, or a mutated luciferase.

In another aspect, the invention features methods of imaging livingcells or animals, e.g., mammals, or humans, that include providing acell expressing a luciferase, or an animal having at least one cellexpressing a luciferase; administering to the cell or animal any one ormore of the luciferins described herein (or an equivalent thereof, e.g.,a salt or ester thereof); and detecting emission therefrom.

Aspects and/or embodiments of the invention can have any one of, orcombinations of, any of the following advantages. Relative tooxyluciferin, the luciferins described herein emit red-shifted lightwhen combined with a suitable luciferase. For example, the luciferinscan emit light having a wavelength of greater than 590 nm, e.g., greaterthan 600 nm, 610 nm, 615 nm, 620, nm, 625 nm, 630 nm, 635 nm, 640 nm,645 nm, 650 nm, 665 nm, or even greater than 675 nm. Emission from theluciferins can be insensitive to pH. While generally the novelluciferins emit light that is red-shifted relative to oxyluciferin, theprecise wavelength emitted can be tuned by selection of the functionalgroups attached to the luciferin. It is expected that the luciferinsdescribed herein will be well tolerated by animals. The luciferins arelikely to be more cell permeable than native luciferin, potentiallyallowing greater access to luciferase within the mouse. Furthermore, thenovel luciferins are likely to have a higher affinity for luciferase,which would allow robust light output under conditions where theluciferin is not present at wild-type luciferin saturatingconcentrations. Modifications in the N-alkyl group(s) could also allowfor modulation of wavelength and light output, leading to luciferinsubstrates with different rates and durations of light output, to bestsuit the imaging experiment. When luciferins are covalently bonded in apocket of a luciferase, fluorescent proteins can be provided. In suchinstances, the chromophore can be protected from the chemicalenvironment in which the fluorescent protein is placed.

“Luciferase” as used herein is an enzyme that operates on a luciferin toproduce light, e.g., near-infrared or visible light. The luciferase canbe wild-type, or it can be a mutated luciferase. An example of aluciferase is wild-type firefly luciferase. A number of suitableluciferases are known in the art (e.g., Branchini et al., Anal. Biochem.(2005) 345:140-148; Nakatsu et al., Nature (2006) 440:372-376; Vivianiet al., Biochemistry (1999) 38:8271).

“Luciferin” as used herein is a material, such as a pure compound ormixture of compounds, that in the presence of a luciferase produceslight. An example of a luciferin is firefly luciferin.

“A near infrared fluorophore” as used herein is one having a maximumemission of greater than 600 nm at physiological pH, e.g., 650 nm orabove.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference herein in their entirety for all that they contain. In case ofconflict, the present specification, including definitions, willcontrol. It is not an admission that any of the information providedherein is prior art or relevant to the presently claimed inventions, orthat any publication specifically or implicitly referenced is prior art.In addition, the materials, methods, and examples are illustrative onlyand not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a series of structures for firefly luciferin (1) and thecorresponding oxyluciferin (represented by (1′A) and (1′B)), thatresults from enzymatic oxidative decarboxylation with firefly luciferase(LUC).

FIG. 2A is a series of generalized structures for some novel aminoluciferins.

FIG. 2B is a series of structures for specific amino luciferins in whichthe nitrogen atom of the amino group is not a member of a ring.

FIGS. 2C and 2D are two series of structures for specific novel aminoluciferins in which the nitrogen atom of the amino group is a member ofone or more rings.

FIGS. 3A-3D are representations of synthetic strategies for makingvarious novel amino luciferins.

FIG. 4A is a series of generalized structures for novel amino luciferinsthat include a NIR acceptor fluorophore at positions 1 and 3.

FIG. 4B is a series of generalized structures for novel aminoluciferins, illustrating specific NIR fluorophores and spacers.

FIG. 4C is a generalized structure for some novel NIRfluorophore-substituted luciferins described herein.

FIG. 5 is a schematic representation of a specific novel amino luciferin(1c) having a mustard group reacting with a thiol group in the pocket of(LUC).

DETAILED DESCRIPTION

Generally, luciferins, or salts or derivatives thereof, e.g., acidesters thereof, are provided that are sized to fit a binding pocket of aluciferase, such as a wild-type or a mutated luciferase.

Luciferins

Generally, and by reference to FIG. 2A, compounds of Structure (I), orsalts or acid esters thereof are provided.

In such compounds of Structure (I), R₁ and R₂ are each independently H,provided that R₁ and R₂ are not both H; a first moiety including up to12 carbon atoms; or a first moiety including a near infraredfluorophore.

The first moiety including up to 12 carbon atoms can also include, e.g.,one or more of N, O, P, S, F, Cl, Br, or I. For example, N can be partof an amino group, an amide group or an imine group. For example, O canbe part of hydroxyl group, a carboxylic acid group, an ester group, ananhydride group, an aldehyde group, a ketone group or an ether group.For example, S can be part of a thio-ester group, a thiol group or athio-ether group. For example, P can be part of a phosphate group, aphosphonate group, a phosphine group, or a phosphoramide group.

For example, the first moiety including up to 12 carbon atoms can be orcan include a hydrocarbon fragment, e.g., an alkyl group, an alkenylgroup, an alkynyl or an aryl group, or a hydrocarbon fragment that issubstituted with one or more of N, O, P, S, F, Cl, Br, or I.

The first moiety can also include a near infrared fluorophore, asdescribed herein.

In compounds of Structure (I), R₃ is H, OH, a second moiety including upto 12 carbon atoms, or a second moiety including a near infraredfluorophore.

The second moiety including up to 12 carbon atoms can be any of thosemoieties described above in reference to R₁ and R₂. The second moietycan also include a near infrared fluorophore, as described herein.

In compounds of Structure (I), R₄ and R₅ are each independently H; OH;or a moiety including up to 6 carbon atoms. The moiety including up to 6carbon atoms can also include, e.g., one or more of N, O, P, S, F, Cl,Br, or I. For example, N can be part of an amino group, an amide group,or an imine group. For example, O can be part of hydroxyl group, acarboxylic acid group, an ester group, an anhydride group, an aldehydegroup, a ketone group or a ether group. For example, S can be part of athio-ester group, a thiol group or a thio-ether group. For example, Pcan be part of a phosphate group, a phosphonate group, a phosphinegroup, or a phosphoramide group. For example, the moiety including up to6 carbon atoms can be or can include a hydrocarbon fragment, e.g., analkyl group, an alkenyl group, an alkynyl or an aryl group, or ahydrocarbon fragment that is substituted with one or more of N, O, P, S,F, Cl, Br, or I.

In compounds of Structure (I), R₆ and R₇ are each independently H, or amoiety including up to 8 carbon atoms. The moiety including up to 8carbon atoms can also include, e.g., one or more of N, O, P, S, F, Cl,Br, or I. For example, N can be part of an amino group, an amide group,or an imine group. For example, O can be part of hydroxyl group, acarboxylic acid group, an ester group, an anhydride group, an aldehydegroup, a ketone group, or a ether group. For example, S can be part of athio-ester group, a thiol group, or a thio-ether group. For example, Pcan be part of a phosphate group, a phosphonate group, a phosphinegroup, or a phosphoramide group. For example, the moiety including up to8 carbon atoms can be or can include a hydrocarbon fragment, e.g., analkyl group, an alkenyl group, an alkynyl or an aryl group, or ahydrocarbon fragment that is substituted with one or more of N, O, P, S,F, Cl, Br, or I.

Referring now also to FIG. 2B, compounds of Structure (I) in which theamino nitrogen is not a member of a ring are exemplified by Structures(1a), (1b), (1c), (1d), (1e) and (1f). Structure (1c) represents areactive N-mustard, which can allow for the luciferin to befunctionalized with a nucleophile-containing moiety, such as compoundbearing an amino or a thiol group. As will be discussed below, such amustard can allow for, e.g., the covalent bonding of the luciferin in abinding pocket, and can enable, e.g., the preparation of fluorescentproteins.

When the amino nitrogen is not a member of a ring, one or more of R₁-R₇can include one or more chiral, or pro-chiral centers.

In compounds of Structure (I), R₁, R₂, R₃, or R₅ may together with oneor more of its immediate neighbors define one or more ring systems, eachincluding up to 14 carbon atoms. For example, the one or more rings canfurther include in a ring or substituted on the ring, e.g., one or moreof N, O, P, S, F, Cl, Br, or I. For example, the balance of the 14carbons atoms not in a ring can substitute a ring, e.g., in the form ofhydrocarbon fragments, e.g., an alkyl group, an alkenyl group, analkynyl or an aryl group, or a hydrocarbon fragment that is substitutedwith one or more of N, O, P, S, F, Cl, Br, or I. For example, N can bepart of an amino group, an amide group or an imine group. For example, Ocan be part of hydroxyl group, a carboxylic acid group, an ester group,an anhydride group, an aldehyde group, a ketone group or a ether group.For example, S can be part of a thio-ester group, a thiol group or athio-ether group.

In preferred embodiments, the one or more ring systems define one ormore 5, 6, and/or 7-membered rings.

Referring back now to FIG. 2A, R₁ and R₂ can together define a ring suchthat the compounds are represented by Structure (II).

In desirable embodiments, R₁ and R₅ can together define a ring such thatthe compounds are represented by Structure (III). Such compounds aredesirable because they have hindered rotation about the Ar—N(R₁R₂) bondof Structure (III), which can enhance red-shifting.

In other desirable embodiments, R₂ and R₃ together define a ring suchthat the compounds are represented by Structure (IV). Such compound arealso desired because they have hindered rotation about the Ar—N(R₁R₂)bond of the of Structure (IV).

In presently preferable embodiments, R₁ and R₅ and R₂ and R₃ togetherdefine a ring such that the compounds are represented by Structure (V).Such compounds are desirable because they have extremely hinderedrotation about the Ar—N(R₁R₂) bond of the of Structure (V).

When the amino nitrogen forms a member of one or more rings, any suchcompounds can include one or more chiral, or pro-chiral centers.

Referring now as well to FIG. 2C, compounds of Structure (I), which arerepresented by Structure (II) are exemplified in by Structures (2a),(2b), (2c), and (2d). Structure (2c) illustrates a compound having aC-based chiral center (marked with *).

Referring now to FIG. 2D, compounds of Structure (I), which arerepresented by Structure (III), (IV) and (V) are exemplified byStructures (3a), (4a-4i) and (5a), respectively. Structures (4g-4i), inwhich the nitrogen atom of the amino group is a member of a 6-membered,unsaturated ring system, and Structure (5a), in which the nitrogen ofthe amino group is a member of two 6-membered rings, each provide forparticularly hindered rotation about the Ar—N(R₁R₂).

The salts of any of the luciferins described herein can be, e.g.,lithium, sodium, potassium, calcium, magnesium or ammonium salts (e.g.,trialkylammonium salts). The esters can be, e.g., NHS esters, alkylesters (e.g., C1-C3 alkyl esters), phenyl esters, benzyl esters oradenosine monophosphate (AMP) esters.

Methods of Making Luciferins

Referring now to FIG. 3A, compounds of Structure (VII′) can be made fromthe chlorobenzothiazole (11) by reacting (11) with a mixture ofHNO₃—H₂SO₄ to produce the corresponding nitro compound, which can bereduced to the amine by treatment of the nitro compound with SnCl₂—H₂Oin DMF. Reductive amination of the amine using R₁CHO and R₂CHO in thepresence of DCE and NaBH(OAc)₃ (see, e.g., JOC (1996) 61:3849) producesthe corresponding substituted amine, and treatment of the substitutedamine with KCN in DMSO gives the corresponding nitrile (see, e.g., Whiteet al., JACS (1966) 88:2015). Treatment of the produced nitrile withD-cysteine and K₂CO₃ in water produces the desired compounds ofStructure (VII'). Alternatively, the Buchwald-Hartwig amination can beused instead of reductive amination (see, e.g., Hartwig, Acc. Chem. Res.(1998), 31, p. 852 and Buchwald et al. Acc. Chem. Res. (1998) 31: 805).

FIGS. 3B-3D show three different methods of making compound (41). FIGS.3B and 3C show methods that start with1-methyl-1,2,3,4-tetrahydroquinolin-6-amine (15), while the method shownin FIG. 3D starts with 2-chlorobenzo[d]thiazol-6-amine (29).

Referring now particularly to FIG. 3B, compound (41) is prepared byfirst treating the hydrochloride salt of compound (15) with sodiumthiosulfate and potassium dichromate in acetic acid to give compound(17) (see, e.g., Tetrahedron 60 (2004) 285-289). Treating compound (17)with formic acid under reflux generates benzothiazole derivative (19)(see, e.g., Tetrahedron 60 (2004) 285-289). Treatment of thebenzothiazole derivative (19) with butyllithium and then TsCN in THFgives the benzothiazole derivative nitrile (21). Treatment of thebenzothiazole derivative nitrile (21) with D-Cys-HCl and NaHCO₃ inwater/methanol generates compound (41).

Referring now particularly to FIG. 3C, in an alternative syntheticmethod (Yarovenko et al., Russ. Chem. Bull. Intl., 51:p 144-147 (2002)),compound (41) is prepared by first treating compound (15) withchloroacetamide, sulfur and TEA in DMF, generating compound (25).Treatment of compound (25) with potassium ferricyanide in aqueous sodiumhydroxide generates compound (27), which is converted to compound (21)by treatment with POCl₃. If desired, potassium ferricyanide in aqueoussodium hydroxide can be replaced by Mn(OAc)₃ in acetic acid or brominein acetic acid. Finally, treatment of the benzothiazole derivativenitrile (21) with D-Cys-HCl and NaHCO₃ in water/methanol generatescompound (41).

Referring now particularly to FIG. 3D, in another synthetic method,compound (40 is prepared by first treating2-chlorobenzo[d]thiazol-6-amine (29) with 3-chloro-propionyl chlorideand sodium bicarbonate to generate the amide derivative (31). Compound(31) is cyclized by treatment with aluminum chloride, and then thecarbonyl is reduced with BH₃-DMS, generating compound (33). Treatment ofcompound (33) with 37% formaldehyde, NaBH(OAc)₃, DCE and acetic acid,generates compound (35). The chloro compound (35) is converted into thecorresponding nitrile (21) by treatment with KCN in DMSO. Finally,treatment of the benzothiazole derivative nitrile (21) with D-Cys-HCland NaHCO₃ in water/methanol generates compound (40.

Luciferins that Include Fluorophores

As described above, in compounds of Structure (I), R₁, R₂ and/or R₃ canbe or can include a fluorophore, e.g., a NIR fluorophore having an amaximum absorption and/or emission of greater than about 600 nm. Such adonor luciferin-acceptor fluorophore configuration can red-shiftemission of the donor luciferin by intramolecular BRET to the acceptorfluorophore. BRET is described in “RED-SHIFTED LUCIFERASE”, U.S.Provisional Patent Application No. 60/904,582, filed on Mar. 2, 2007,and U.S. patent application Ser. No. 12/040,797 by the same inventor,both of which are incorporated herein by reference in their entirety.

In some embodiments, the acceptor fluorophore has a maximum emission ofgreater than 605 nm, e.g., greater than 610 nm, 615 nm, 620, nm, 625 nm,630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 665 nm, or even greater than 675nm.

In some instances, the fluorophore is directly bonded to the amino groupof the luciferin, and in other instances, the fluorophore is furtherremoved by a spacer.

FIG. 4A shows a luciferin that includes a donor portion and an acceptorportion bonded to the amino group of the luciferin by a spacer (VIII),and a luciferin that includes a donor portion and an acceptor portionbonded to the aryl ring of the luciferin at the ortho position by aspacer (X).

In some embodiments, the spacer includes up to 24 carbon atoms.Optionally, the spacer can also include one or more N, O, P, S, F, Cl,Br, or I. For example, N can be part of an amino group, an amide groupor an imine group. For example, O can be part of a hydroxyl group, acarboxylic acid group, an ester group, an anhydride group, an aldehydegroup, a ketone group or a ether group. For example, S can be part of athio-ester group, or a thiol group of a thio-ether group. For example,the 24 carbon atoms can be or can include a hydrocarbon fragment, e.g.,an alkyl group, an alkenyl group, an alkynyl or an aryl group, or ahydrocarbon fragment that is substituted with one or more of N, O, S, F,Cl, Br, or I.

Referring now to FIG. 4B, in particular embodiments, the luciferin isrepresented by structure (XI). In such instances, the spacer can an C10alkyl amide spacer, and the fluorophores can be any one of (11a), (11b),(11e), (11d) or (11e).

In some embodiments, the spacer is polymeric, e.g., a polymer fragmentof a water-soluble polymer. For example, the spacer can be apolyethylene glycol (PEG) moiety or a PEG copolymer moiety, e.g.,defined by —(CH₂CH₂O)_(n)—, in which n is an integer from 1 to 5.

Referring again to FIG. 4B, in particular embodiments, the luciferin isrepresented by structure (XII). In such instances, the spacer can be aPEG moiety in which n (see above) is between about 1 and 250. Just aswas the case with Structure (XII), the fluorophores can be, e.g., areany one of (11a), (11b), (11e), (11d) or (11e) or others.

Even other structures are possible. For example and by reference to FIG.4C, other luciferins having dangling fluorophores can take the form ofStructure (XIII). In such compounds of Structure (XIII), A is OH (whichcorresponds to a native firefly luciferin-type structure) or NR₁₀R₁₁,R₁₀ and R₁₁ each being independently H, or a third moiety that includesup to 12 carbon atoms. R₁₂ is a third moiety that includes a nearinfrared fluorophore, R₁₃ and R₁₄ are each independently H, OH, or asecond moiety comprising up to 6 carbon atoms, and R₁₅ and R₁₆ are eachindependently H, or a second moiety that includes up to 8 carbon atoms.R₁₀, R₁₁, R₁₂, and R₁₄ together with one or more of its immediateneighbors may define one or more ring systems, each including up to 14carbon atoms. The third moiety including up to 12 carbon atoms can beany of those moieties described above in reference to R₁ and R₂. Thesecond moiety that includes up to 6 carbon atoms can be any of thosemoieties described above in reference to R₄ and R₅. The second moietythat includes up to 8 carbon atoms can be any of those moietiesdescribed above in reference to R₆ and R₇.

Fluorescent Proteins

When luciferins are covalently bonded in a pocket of a luciferase,fluorescent proteins are generated. In such instances, the luciferinchromophore can be protected from the chemical environment in which thefluorescent protein is placed.

Referring now to FIG. 5, the mustard compound (1c), or optionally theaziridine compound (2a) of FIG. 2C, can be covalently bonded in aluciferase pocket by reacting the compound (1c or 2a) with a luciferasehaving an exposed nucleophile, such as a thiol group or an amino group.FIG. 5 shows the mustard compound (1c) reacting with a thiol group in apocket of a luciferase.

Mutated Luciferases

The red-shifted luciferins described herein may, in some cases, be toolarge to fit inside the binding pocket of a wild-type luciferase (forthe crystal structure of a wild-type luciferase and several mutants seeNakatsu et al., Nature (2006) 440:372-376). In this case, the bindingpocket of the luciferase can be mutated to enlarge the pocket toaccommodate the red-shifted luciferin (for luciferase mutants see, e.g.,Branchini et al., Anal Biochem, 2005, 345(1):140 and Branchini et al.,Biochem. 42 (2003) 10429-36). For example, various amino acids can bealtered, e.g., using standard site-directed mutagenesis methods, toamino acids with smaller side chains, thereby enlarging the bindingpocket. For example, for substrate accommodation, amino acid residuescorresponding to Arginine 218, Isoleucine 286, Serine 284, Serine 314,Alanine 313, Threonine 343 and/or Serine 347 of firefly luciferase canbe altered. For example, for accessibility to solvent (e.g., fluorophoretethering), amino acid residues corresponding to Glutamate 311, Arginine337 and/or Asparagine 229 of firefly luciferase can be altered. Ingeneral, conservative substitutions will be desirable. Mutatedluciferases can be evaluated for activity using a standard luciferaseassay, e.g., by contacting the mutated luciferase with a wild-typeluciferin or a red-shifted luciferin as described herein, and detectingphotons emitted therefrom.

For example, rational design methods can be used in which empiricallydetermined or computationally generated models of a luciferin describedherein and a luciferase binding site are evaluated, and mutationsselected that optimize the fit of the luciferin in the binding site ofthe luciferase. Alternatively, a panel of binding site mutants ofluciferase can be produced, and standard assays, e.g., high-throughputscreening, can be used to identify optimal luciferin-mutant luciferasepairs for a particular application.

Methods of Use

The methods and compositions described herein can be used for in vivoimaging and may improve the speed, detection limit, and depthpenetration of bioluminescence imaging. For example, the methodsdescribed herein can be used for the rapid and inexpensive evaluation oftumor progression and response to anti-cancer therapeutics in smallanimals, e.g., using transgenic non-human animals, e.g., mice, thatexpress a luciferase reporter gene linked to a promoter or gene that isexpressed, e.g., selectively expressed, in the cells that are desired tobe imaged (Greer and Szalay, Luminescence, 2002, 17:43-74). In the sameway, expression of a selected protein of interest can be imaged in realtime in a living cell or animal, using a cell or transgenic animal thatexpresses a reporter construct including a nucleic acid encoding aluciferase linked in frame to a nucleic acid encoding the selectedprotein of interest, or to the promoter for the selected protein.

In general, the methods will be performed on cells or animals (e.g.,non-human mammals, e.g., experimental animals, such as rodents, e.g.,rats or mice) that express a luciferase or a mutated luciferase reporterconstruct. One of skill in the art will readily be able to make suchcells or animals using standard molecular biological techniques.Sufficient amounts of any of the modified luciferins described hereinare then added or administered to the cells or animals, and images ofthe NIR bioluminescence obtained using standard imaging methods. In thisway, promoter activity, protein expression, protein subcellularlocalization, protein translocation, and protein half-life, can beevaluated in real time in living cells and animals.

When an experimental animal is used, the cells containing the NIRbioluminescence can be identified and excised, and evaluated further,e.g., using assays for gene expression, protein expression, or othergenetic or biochemical parameters.

The design of specific luciferin/luciferase pairs (e.g., with differentemission maxima) can allow for simultaneous imaging of bioluminescencefrom two or more luciferases.

Imaging Methods

The methods described herein can be practiced with any imaging systemthat can detect near infrared bioluminescence, e.g., the in vivo imagingsystems described in Doyle et al., Cellular Microbiology (2004)6(4):303-317. Common imaging systems are available from Xenogen (e.g.,IVIS), Hamamatsu, Roper, and Kodak.

EXAMPLES

The disclosure is further described in the following examples, which donot limit its scope.

Example 1 Synthesis of Dimethylamino-Luciferin (1a)

This Example describes the synthesis of dimethylamino-luciferin (1a)(structure shown below).

6-amino-2-chlorobenzothiazole (29, FIG. 3D)

6-amino-2-chlorobenzothiazole (29) was synthesized following theprocedure of White et al., 1966 (JACS 88, 2015).

2-chloro-6-dimethylaminobenzothiazole (40) (Shown Below)

To a solution of (29) (0.1 mmol, 18.5 mg) in 1 ml dichloroethane wasadded 90 μl 37% formaldehyde, followed by 63.6 mg sodiumtriacetoxyborohydride, and 12 μl acetic acid. After 2 h, the solutionwas poured into water and extracted with ethyl acetate. The combinedextracts were washed with water, dried over sodium sulfate, and thesolvent removed by rotary evaporation. The crude material (21 mg)contained a small amount of monomethylated product, but was sufficientlypure to use directly in the next step. ¹H-NMR (CDCl₃): d 7.75 (d, 1H,J=9.2 Hz), 6.95 (d, 1H, J=2.4 Hz), 6.90 (dd, 1H, J=2.4, 9.2 Hz), 3.02(s, 6H).

2-cyano-6-dimethylaminobenzothiazole (44) (Shown Below)

Potassium cyanide (18 mg) and (40) (18 mg) were dissolved in 1 ml DMSOand heated to 120° C. for 4 h. After cooling to room temperature, thereaction was poured into 0.2M potassium phosphate, pH 4.5. The aqueouslayer was extracted with ethyl acetate three times, washed with water,and dried over sodium sulfate. The solvent was removed by rotaryevaporation and the residue purified by silica gel chromatography (20%ethyl acetate in hexanes). ¹H-NMR (d₆-DMSO): d 7.97 (d, 1H, J=9.2 Hz),7.38 (d, 1H, J=2.4 Hz), 7.17 (dd, 1H, J=2.4, 9.2 Hz), 3.03 (s, 6H).

D-2-(6′-dimethylamino-2′-benzothiazolyl)-Δ²-thiazoline-4-carboxylic acid(1a)

D-cysteine-HCl (2.5 mg) was dissolved in 0.6 ml of deoxygenated 50 mMpotassium phosphate buffer, pH 8.1 and added to 2 mg (10 μmol) of (44)in 0.6 ml of deoxygenated methanol and stirred under argon at roomtemperature. After 2 h, the methanol was removed by rotary evaporation,and the remaining aqueous solution was extracted with ethyl acetatetwice and dried with sodium sulfate. Removal of the solvent by rotaryevaporation yielded 3 mg of an orange-red solid. ¹H-NMR (d₆-DMSO): d7.89 (d, 1H, J=9.2 Hz), 7.29 (d, 1H, J=2.4 Hz), 7.05 (dd, 1H, J=2.4, 9.2Hz), 5.35 (app t, 1H, X of ABX, J=8-10 Hz), 3.74-3.59 (m, 2H, AB ofABX), 3.01 (s, 6H).

Example 2 Synthesis of Monoethylamino-Luciferin2-chloro-6-ethylaminobenzothiazole

To a solution of (29) (0.2 mmol, 36.8 mg) in 2 ml dichloroethane wasadded 0.2 mmol acetaldehyde, followed by 63.6 mg sodiumtriacetoxyborohydride, and 12 μl acetic acid. After 2 h, the solutionwas poured into water and extracted with ethyl acetate. The combinedextracts were washed with water, dried over sodium sulfate, and thesolvent removed by rotary evaporation.

2-cyano-6-ethylaminobenzothiazole

Potassium cyanide (18 mg) and 2-chloro-6-ethylaminobenzothiazole (18 mg)were dissolved in 1 ml DMSO and heated to 120° C. for 4 h. After coolingto room temperature, the reaction was poured into 0.2M potassiumphosphate, pH 4.5. The aqueous layer was extracted with ethyl acetatethree times, washed with water, and dried over sodium sulfate. Thesolvent was removed by rotary evaporation and the residue purified bysilica gel chromatography (20% ethyl acetate in hexanes). ¹H-NMR(d₆-DMSO): δ 7.85 (d, 1H, J=9.2 Hz), 7.13 (d, 1H, J=2.4 Hz), 6.98 (dd,1H, J=2.4, 8.8 Hz), 6.44 (br t, 1H), 3.09 (m, 2H), 1.18 (t, 3H, J=7.2Hz).

D-2-(6′-ethylamino-2′-benzothiazolyl)-Δ²-thiazoline-4-carboxylic acid

D-cysteine-HCl (7 mg, 40 pimp was dissolved in 1 ml of deoxygenated 50mM potassium phosphate buffer, pH 8 and added to 6.3 mg (31 μmol) of2-cyano-6-ethylaminobenzothiazole in 1 ml of deoxygenated methanol andstirred under argon at room temperature. After 2 h, the solution wasacidified to pH 5 with 0.1M HCl and the methanol was removed by rotaryevaporation. The precipitated solid and the remaining aqueous solutionwas extracted with ethyl acetate three times and dried with sodiumsulfate. Removal of the solvent by rotary evaporation yielded 8.8 mg ofan orange-red solid. ¹H-NMR (d₆-DMSO): δ 7.76 (d, 1H, J=8.8 Hz), 7.03(s, 1H), 6.86 (d, 1H, J=9.2 Hz), 6.35 (m, 1H), 5.32 (app t, 1H, X ofABX), 3.75-3.55 (m, 2H, AB of ABX), 3.09 (m, 2H), 1.18 (t, 3H, J=7.2Hz).

Example 3 Synthesis of Monoisopropylamino-Luciferin2-chloro-6-isopropylaminobenzothiazole

To a solution of (29) (0.2 mmol, 36.8 mg) in 2 ml dichloroethane wasadded 0.2 mmol acetone (14.5 followed by 63.6 mg sodiumtriacetoxyborohydride, and 12 μl acetic acid. After 2 h, the solutionwas poured into water and extracted with ethyl acetate. The combinedextracts were washed with water, dried over sodium sulfate, and thesolvent removed by rotary evaporation.

2-cyano-6-isopropylaminobenzothiazole

Potassium cyanide (18 mg) and 2-chloro-6-isopropylaminobenzothiazole (18mg) were dissolved in 1 ml DMSO and heated to 120° C. for 4 h. Aftercooling to room temperature, the reaction was poured into 0.2M potassiumphosphate, pH 4.5. The aqueous layer was extracted with ethyl acetatethree times, washed with water, and dried over sodium sulfate. Thesolvent was removed by rotary evaporation and the residue purified bysilica gel chromatography (20% ethyl acetate in hexanes). ¹H-NMR(d₆-DMSO): δ 7.83 (d, 1H, J=9.2 Hz), 7.15 (d, 1H, J=2.4 Hz), 6.97 (dd,1H, J=2.4, 8.8 Hz), 6.52 (bd, 1H, J=7.2 Hz), 3.61 (m, 1H), 1.15 (d, 6H,J=6 Hz).

D-2-(6′-isopropylamino-2′-benzothiazolyl)-Δ²-thiazoline-4-carboxylicacid

D-cysteine-HCl (8.8 mg, 50 pimp was dissolved in 1.25 ml of deoxygenated50 mM potassium phosphate buffer, pH 8 and added to 9.1 mg (42 μmol) of2-cyano-6-isopropylaminobenzothiazole in 1.25 ml of deoxygenatedmethanol and stirred under argon at room temperature. After 2 h, thesolution was acidified to pH 5 with 0.1M HCl and the methanol wasremoved by rotary evaporation. The precipitated solid and the remainingaqueous solution was extracted with ethyl acetate three times and driedwith sodium sulfate. Removal of the solvent by rotary evaporationyielded 15 mg of an orange-red solid. ¹H-NMR (d₆-DMSO): δ 7.75 (d, 1H,J=9.2 Hz), 7.05 (d, 1H, J=2.4 Hz), 6.85 (dd, 1H, J=2.4, 8.8 Hz), 6.22(bd, 1H, J=7.2 Hz), 5.32 (app t, 1H, X of ABX, J=8-10 Hz), 3.61 (m, 1H),3.55-3.72 (m, 2H, AB of ABX), 1.14 (d, 6H, J=6.4 Hz).

Example 4 Synthesis of Mono-N-Butylamino-Luciferin2-chloro-6-butylaminobenzothiazole

To a solution of (29) (0.2 mmol, 36.8 mg) in 2 ml dichloroethane wasadded 0.2 mmol butyraldehyde (18 followed by 63.6 mg sodiumtriacetoxyborohydride, and 12 μl acetic acid. After 2 h, the solutionwas poured into water and extracted with ethyl acetate. The combinedextracts were washed with water, dried over sodium sulfate, and thesolvent removed by rotary evaporation, yielding 44.2 mg of a yellowsolid. ¹H-NMR (CDCl₃): δ 7.67 (d, 1H, J=9.2 Hz), 6.84 (d, 1H, J=2.4 Hz),6.72 (dd, 1H, J=2.4, 9.2 Hz), 3.35 (bt, 1H), 3.15 (t, 2H), 1.65 (m, 2H),1.45 (m, 2H), 0.97 (t, 3H, J=7.6 Hz).

2-cyano-6-butylaminobenzothiazole

Potassium cyanide (18 mg) and 2-chloro-6-butylaminobenzothiazole (18 mg)were dissolved in 1 ml DMSO and heated to 120° C. for 4 h. After coolingto room temperature, the reaction was poured into 0.2M potassiumphosphate, pH 4.5. The aqueous layer was extracted with ethyl acetatethree times, washed with water, and dried over sodium sulfate. Thesolvent was removed by rotary evaporation and the residue purified bysilica gel chromatography (20% ethyl acetate in hexanes). ¹H-NMR(d₆-DMSO): δ 7.84 (d, 1H, J=8.8 Hz), 7.14 (d, 1H, J=2.4 Hz), 6.99 (dd,1H, J=2.4, 9.2 Hz), 6.67 (br t, 1H), 3.06 (m, 2H), 1.55 (m, 2H), 1.39(m, 2H), 0.90 (t, 3H, J=7.4 Hz).

D-2-(6′-butylamino-2′-benzothiazolyl)-Δ²-thiazoline-4-carboxylic acid

D-cysteine-HCl (7 mg, 40 mmol) was dissolved in 1 ml of deoxygenated 50mM potassium phosphate buffer, pH 8 and added to 6.6 mg (29 mmol) of2-cyano-6-butylaminobenzothiazole in 1 ml of deoxygenated methanol andstirred under argon at room temperature. After 2 h, the solution wasacidified to pH 5 with 0.1M HCl and the methanol was removed by rotaryevaporation. The precipitated solid and the remaining aqueous solutionwas extracted with ethyl acetate three times and dried with sodiumsulfate. Removal of the solvent by rotary evaporation yielded 8.5 mg ofan orange-red solid. ¹H-NMR (d₆-DMSO): δ 7.75 (d, 1H, J=9.2 Hz), 7.03(s, 1H), 6.87 (d, 1H, J=9.2 Hz), 6.36 (m, 1H), 5.32 (app t, 1H, X ofABX), 3.75-3.55 (m, 2H, AB of ABX), 3.04 (m, 2H), 1.54 (m, 2H), 1.37 (m,2H), 0.90 (t, 3H, J=7.2 Hz).

Example 5 Luciferase Assay

Firefly luciferase (from Promega's pGL2-Basic vector (X65323), orpGL3-Basic vector (U47295)) was cloned into pGEX-6P-1 and expressed as aGST fusion protein in E. coli. The GST tag was removed by cleavage withPreScission™ protease, providing a composition of substantially purifiedluciferase. The emission spectrum of luciferase at pH 7.8 was recordedin a Spex Fluoromax™-3. The maximal emission in the presence of thenative substrate D-luciferin was 555 nm. With amino-luciferin assubstrate, the emission shifted to 593 nm (White et al., JACS (1966) 88:2015 reported 605 nm). The monoalkylaminoluciferins (ethylamino,isopropylamino, and n-butylamino) all shifted emission to 607 nm. Whendimethylamino-luciferin (1a) was used as a substrate, the emission wasbathochromatically shifted to 624 nm.

OTHER EMBODIMENTS

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made without departing fromthe spirit and scope of the invention. Other embodiments are within thescope of the following claims.

1. A method of imaging a sample, the method comprising: obtaining asample comprising a luciferase; contacting the sample with a compound ofStructure (III) or (IV), or a salt or acid ester thereof:

wherein: R₁ and R₂ are each independently H, C₁-₁₂ alkyl optionallysubstituted by an amino group, an amide group, an imine group, ahydroxyl group, a carboxylic acid group, an ester group, an anhydridegroup, an aldehyde group, a ketone group, an ether group, a thio-estergroup, a thiol group, a thioether group, a phosphate group, aphosphonate group, a phosphine group, a phosphoramide group, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, I,or a near infrared fluorophore, optionally with a spacer, provided thatR₁ and R₂ are not both H; R₃ is H, OH, C₁-₁₂ alkyl optionallysubstituted by an amino group, an amide group, an imine group, ahydroxyl group, a carboxylic acid group, an ester group, an anhydridegroup, an aldehyde group, a ketone group, an ether group, a thio-estergroup, a thiol group, a thioether group, a phosphate group, aphosphonate group, a phosphine group, a phosphoramide group, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, I,or a near infrared fluorophore, optionally with a spacer; R₄ and R₅ areeach independently H, OH, or C₁-₆ alkyl optionally substituted by anamino group, an amide group, an imine group, a hydroxyl group, acarboxylic acid group, an ester group, an anhydride group, an aldehydegroup, a ketone group, an ether group, a thio-ester group, a thiolgroup, a thioether group, a phosphate group, a phosphonate group, aphosphine group, a phosphoramide group, an alkyl group, an alkenylgroup, an alkynyl group, an aryl group, F, Cl, Br, or I; R₆ and R₇ areeach independently H, or C₁-₈ alkyl optionally substituted by an aminogroup, an amide group, an imine group, a hydroxyl group, a carboxylicacid group, an ester group, an anhydride group, an aldehyde group, aketone group, an ether group, a thio-ester group, a thiol group, athioether group, a phosphate group, a phosphonate group, a phosphinegroup, a phosphoramide group, an alkyl group, an alkenyl group, analkynyl group, an aryl group, F, Cl, Br, or I; R₁, R₂, R₃, or R₅together with one or more of its immediate neighbors define one or more5, 6, or 7-membered rings; wherein the rings optionally include one ormore groups selected from: an amino group, an amide group, an iminegroup, a hydroxyl group, a carboxylic acid group, an ester group, ananhydride group, an aldehyde group, a ketone group, an ether group, athio-ester group, a thiol group, a thioether group, a phosphate group, aphosphonate group, a phosphine group, a phosphoramide group, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, andI; and the spacer is C₁-₂₄ alkyl optionally substituted by one or moregroups selected from: an amino group, an amide group, an imine group, ahydroxyl group, a carboxylic acid group, an ester group, an anhydridegroup, an aldehyde group, a ketone group, an ether group, a thio-estergroup, a thiol group, a thioether group, a phosphate group, aphosphonate group, a phosphine group, a phosphoramide group, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, andI; and detecting bioluminescent emission from the sample.
 2. The methodof claim 1, wherein the spacer is a polymer fragment of a water-solublepolymer fragment.
 3. The method of claim 1, wherein R₃, R₄, R₅, R₆ andR₇ are each hydrogen, or an alkyl group having fewer than 4 carbonatoms.
 4. A method of imaging a sample, the method comprising: obtaininga sample comprising a luciferase; contacting the sample with a compoundof Structure (V), or salts or acid esters thereof:

 wherein R₄ is H, OH, or C₁-₆ alkyl optionally substituted by an aminogroup, an amide group, an imine group, a hydroxyl group, a carboxylicacid group, an ester group, an anhydride group, an aldehyde group, aketone group, an ether group, a thio-ester group, a thiol group, athioether group, a phosphate group, a phosphonate group, a phosphinegroup, a phosphoramide group, an alkyl group, an alkenyl group, analkynyl group, an aryl group, F, Cl, Br, or I; R₆ and R₇ are eachindependently H or C₁-₈ alkyl optionally substituted by an amino group,an amide group, an imine group, a hydroxyl group, a carboxylic acidgroup, an ester group, an anhydride group, an aldehyde group, a ketonegroup, an ether group, a thio-ester group, a thiol group, a thioethergroup, a phosphate group, a phosphonate group, a phosphine group, aphosphoramide group, an alkyl group, an alkenyl group, an alkynyl group,an aryl group, F, Cl, Br, or I; R₁ and R₅ and R₂ and R₃ together withone or more of its immediate neighbors define one or more 5, 6, or7-membered rings; and wherein the rings optionally include one or moregroups selected from: an amino group, an amide group, an imine group, ahydroxyl group, a carboxylic acid group, an ester group, an anhydridegroup, an aldehyde group, a ketone group, an ether group, a thio-estergroup, a thiol group, a thioether group, a phosphate group, aphosphonate group, a phosphine group, a phosphoramide group, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, andI; and, detecting bioluminescent emission from the sample.
 5. The methodof claim 1, wherein R₁ and/or R₂ are each C₁-₁₂ alkyl linked to a nearinfrared fluorophore.
 6. The method of claim 1, wherein R₃ is C₁-₁₂alkyl linked to a near infrared fluorophore.
 7. The method of claim 1,wherein the compounds are selected from:


8. The method of claim 1, wherein the luciferase has a mutation atSerine 347 to an amino acid with a smaller side chain.
 9. The method ofclaim 8, wherein the luciferase has a mutation at Serine 347 to alanine10. The method of claim 4, wherein the sample comprises a cell lysate.11. The method of claim 4, further comprising providing a plurality ofsamples, and detecting emissions from each of the samples.
 12. A methodof imaging an animal, the method comprising: obtaining an animal havingat least one cell expressing a luciferase; administering to the animal acompound of Structure (V), or salts or acid esters thereof:

 wherein R₄ is H, OH, or C₁-₆ alkyl optionally substituted by an aminogroup, an amide group, an imine group, a hydroxyl group, a carboxylicacid group, an ester group, an anhydride group, an aldehyde group, aketone group, an ether group, a thio-ester group, a thiol group, athioether group, a phosphate group, a phosphonate group, a phosphinegroup, a phosphoramide group, an alkyl group, an alkenyl group, analkynyl group, an aryl group, F, Cl, Br, or I; R₆ and R₇ are eachindependently H or C₁-₈ alkyl optionally substituted by an amino group,an amide group, an imine group, a hydroxyl group, a carboxylic acidgroup, an ester group, an anhydride group, an aldehyde group, a ketonegroup, an ether group, a thio-ester group, a thiol group, a thioethergroup, a phosphate group, a phosphonate group, a phosphine group, aphosphoramide group, an alkyl group, an alkenyl group, an alkynyl group,an aryl group, F, Cl, Br, or I; R₁ and R₅ and R₂ and R₃ together withone or more of its immediate neighbors define one or more 5, 6, or7-membered rings; and wherein the rings optionally include one or moregroups selected from: an amino group, an amide group, an imine group, ahydroxyl group, a carboxylic acid group, an ester group, an anhydridegroup, an aldehyde group, a ketone group, an ether group, a thio-estergroup, a thiol group, a thioether group, a phosphate group, aphosphonate group, a phosphine group, a phosphoramide group, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, andI; and, detecting emission from the compound to image the animal.
 13. Amethod of imaging a cell, the method comprising: obtaining a cellexpressing a luciferase; contacting the cell with a compound ofStructure (V), or salts or acid esters thereof:

 wherein R₄ is H, OH, or C₁-₆ alkyl optionally substituted by an aminogroup, an amide group, an imine group, a hydroxyl group, a carboxylicacid group, an ester group, an anhydride group, an aldehyde group, aketone group, an ether group, a thio-ester group, a thiol group, athioether group, a phosphate group, a phosphonate group, a phosphinegroup, a phosphoramide group, an alkyl group, an alkenyl group, analkynyl group, an aryl group, F, Cl, Br, or I; R₆ and R₇ are eachindependently H or C₁-₈ alkyl optionally substituted by an amino group,an amide group, an imine group, a hydroxyl group, a carboxylic acidgroup, an ester group, an anhydride group, an aldehyde group, a ketonegroup, an ether group, a thio-ester group, a thiol group, a thioethergroup, a phosphate group, a phosphonate group, a phosphine group, aphosphoramide group, an alkyl group, an alkenyl group, an alkynyl group,an aryl group, F, Cl, Br, or I; R₁ and R₅ and R₂ and R₃ together withone or more of its immediate neighbors define one or more 5, 6, or7-membered rings; and wherein the rings optionally include one or moregroups selected from: an amino group, an amide group, an imine group, ahydroxyl group, a carboxylic acid group, an ester group, an anhydridegroup, an aldehyde group, a ketone group, an ether group, a thio-estergroup, a thiol group, a thioether group, a phosphate group, aphosphonate group, a phosphine group, a phosphoramide group, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, F, Cl, Br, andI; and, detecting emission from the compound to image the cell.
 14. Themethod of claim 13, wherein the luciferase is expressed from a reporterconstruct.
 15. The method of claim 14, wherein the reporter constructcomprises a nucleic acid encoding a luciferase operably linked to aselected promoter, and detecting emission from the luciferin provides anindication of promoter activity.
 16. The method of claim 14, wherein thereporter construct comprises a nucleic acid encoding a luciferase linkedin frame to a nucleic acid encoding a selected protein, and detectingemission from the luciferin provides an indication of one or more ofexpression, subcellular localization, translocation, and half-life ofthe protein.